Sepsis remains a devastating disease exacting substantial mortality\morbidity and consuming significant health care resources. Hydrocortisone, Ascorbic acid and Thiamine (HAT) therapy has been hypothesized to reduce cell and organ injury through synergistic actions. A small clinical HAT trial improved survival in septic patients and sparked interest into examining mechanism(S). Pre-clinical models have limitations and flawed murine models have not produced effective clinical therapies. These deficiencies include use of endotoxin injections, failure to provide routine, effective sepsis treatments (antibiotics and fluids) and very high mortality rates. However, appropriate sepsis models do respond to proven sepsis treatments and have predicted failed interventions. The premise for the current proposal derives from the hypothesis that the three HAT components synergize to reduce cell and organ injury, especially in the kidney. The current R21 proposal addresses the unresolved issues of defining how HAT therapy is effective through two specific aims. The first aim will measure in vivo reductions in oxidative stress (OS) and improvements in phagocytic cell function. Non-invasive physiologic measurements of heart rate (HR) obtained six hours after the onset of sepsis accurately predict mortality in the cecal ligation and puncture (CLP) model. Outbred mice, both males and females, will be stratified into predicted to live or die based on HR and then randomized to receive HAT or vehicle, an experimental approach with both innovation and significance. The design is innovative since it directs the therapy towards the animals where it may be beneficial, rather than employing the typical one-size-fits-all approach. The plan is significant, since clinical trials could adopt the same approach of stratifying patients. We will measure multiple OS parameters in several different organs to determine if HAT reduces OS in septic mice. Such studies are not really feasible in patients since actual tissue samples are required, and sampling of vital organs such as the kidney would be difficult to do in a septic patient. OS will be quantified in these initial studies as a marker of efficacy, but the premise for the proposal is that all three HAT components synergize to reduce cell injury and not merely reduce OS. Phagocytic cell function has been postulated to be decreased by OS in sepsis and these cells will be studied with innovative techniques in aim 1. Alternative treatments with double therapy combinations will be tested, as well as different doses and times of treatment after the onset of sepsis. Since renal injury is the most frequently observed dysfunctional organ in sepsis, the second specific aim examines the mechanism(s) of how HAT reduces this injury. Sophisticated in vivo techniques will localize hypoxia-stressed individual cells, and mitochondrial function will quantified to determine if HAT improves this cellular powerhouse. Successful completion of the proposed studies provides a foundation for understanding the mechanisms of improved cell and organ function. The data from this R21 grant will be used to support future studies to identify pathways driving sepsis induced organ injury.